Project Summary: The goal of this proposal is to test the hypothesis that TGF-?1 released from platelets and activated by shear stress in the circulation contributes to the pathophysiology of aortic stenosis (AS) and the complications associated with implantation of left ventricular assist device (LVAD) in heart failure (HF) patients. This will build upon our previous and new preliminary studies demonstrating that: 1) shear force can activate latent TGF-?1, 2) thiol-disulfide exchange contributes to the activation process, 3) mice deficient in platelet TGF-?1 are protected from developing cardiac fibrosis in response to pressure overload, 4) Reversa mice develop AS and increased plasma TGF-?1 levels that correlate with shear stress, and 4) both AS and LVAD-implanted HF patients have increased levels of TGF-?1 compared to controls. We will extend our findings by integrating data from biochemical analyses, animal models, and human patient studies. Aim 1 is to test the hypothesis that shear activates TGF-?1 by facilitating thiol-disulfide exchange involving TGF-?1, thiol isomerases, and/or other thiol-containing proteins, such as TSP1 and vWf. Purification of TGF-?1 and other thiol-containing proteins from platelets, proteomic analysis using mass spectrometry, and mutational studies of recombinant proteins will be employed to identify the shear-induced thiol-disulfide exchange responsible for TGF-?1 activation. Aim 2 will test the hypothesis that TGF-?1 is released from platelets and subsequently activated by high shear in vivo using two mouse models: a surgically-induced Ascending Aorta Constriction model to acutely simulate high shear, and a Reversa mouse model that spontaneously develops AS and high shear across the aortic valve. We will assess TGF-?1 release and activation as well as the impact on TGF-?1 activation in mice with a mutation in the latency-associated protein (LAPC33S), whose TGF-?1 cannot be activated by shear and in mice selectively deficient in platelet PDI (PDIflox). Reversa mice have increased levels of TGF-?1 that correlates with shear stress. We will test the role of platelet TGF-?1 and PDI on AS progression by breeding Reversa mice with platelet-specific PF4Cre/Tgfb1flox and PF4Cre/PDIflox mice. A TGF-?1 neutralizing antibody will be used to monitor AS progression as a model for intervention. Aim 3 will test the hypothesis that release and activation of TGF-?1 occurs in vivo by assessment of TGF-?1 levels in AS and LVAD-implanted HF patients. AS patients have elevated TGF-?1 levels compared to controls. TGF-?1 levels will be correlated with AS valve pathophysiology. Whether TGF-?1 levels are decreased after valve replacement surgery will be assessed. TGF-?1 levels are elevated in LVAD-implanted HF patients and associated with the loss of vWf multimers. We will monitor TGF-?1 levels through heart transplantation and correlate with vWf multimers to establish TGF-?1 and vWf-multimers as surrogate markers for the risk of thrombosis and hemorrhage, respectively. Collectively, these studies will illuminate and guide development of potential interventions to prevent AS progression as well as diagnostic tools to detect early complications in post-LVAD implantation HF patients.